Title: Development of Affordable Bioelectronic Interfaces Using Medically Relevant Soluble Enzymes
1Development of Affordable Bioelectronic
Interfaces Using Medically Relevant Soluble
Enzymes
2006 AIChE Annual Meeting San Francisco, CA
- Brian L. Hassler1, Maris Laivenieks2, Claire
Vieille2, J. Gregory Zeikus2, and Robert M.
Worden1
1-Department of Chemical Engineering and
Materials Science 2-Department of Biochemistry
and Molecular Biology Michigan State
University, East Lansing, Michigan
2Presentation Outline
- Motivation
- Dehydrogenase enzymes
- Formation of bioelectronic interfaces
- Characterization techniques
- Results
- Summary
3Motivation
- Rapid detection
- Identification of multiple analytes
- High throughput screening
- Affordable fabrication
4Dehydrogenase Enzymes
- Catalyze electron transfer reactions
- Cofactor dependence NAD(P)
- Challenge cofactor recycling
5Enzyme Interface Assembly
- Cysteine branched, trifunctional linker
- Thiol group self assembles on gold
- Carboxyl group binds to electron mediator
- Amine group binds to cofactor
- Mediator used
- Toluidine Blue O (TBO)
6Reaction Mechanism
N-Hydroxysulfosuccinimide
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide
Hassler et al., Biosensors and Bioelectronics,
21(11), 2146-2154 (2006)
7Presentation Outline
- Motivation
- Sensing mechanisms
- Formation of bioelectronic interfaces
- Characterization techniques
- Results
- Summary
8Chronoamperometry
- Technique
- Step change in potential
- Measure current vs. time
- Parameters obtained
- Electron transfer coefficients (ket)
- Charge (Q)
- Surface coverage (?)
Zayats et al., Journal of the American Chemical
Society, 124, 14724-15735 (2002)
Katz, E. and I. Willner, Langmuir, 13(13),
3364-3373 (1997)
9Cyclic Voltammetry
- Technique
- Conduct potential sweep
- Measure current
- Parameters obtained
- Sensitivity (slope)
- Maximum turnover (TRmax)
10Constant Potential Amperometry
- Technique
- Set constant potential
- Vary analyte concentration
- Parameters obtained
- Sensitivity
11Presentation Outline
- Motivation
- Sensing mechanisms
- Formation of bioelectronic interfaces
- Characterization techniques
- Results
- Summary
12The Current System
- Protein array
- 4 working electrodes
- Diameter 3 mm
- Counter electrode
- Electrode formation
- Reservoir in PDMS
- Molecular self-assembly
- Different enzymes
Polydimethylsiloxane (PDMS)
13Sorbitol Dehydrogenase (SDH)
- Organism Pseudomonas sp. KS-E1806
- Cofactor dependence NAD
- Temperature stability 30-50?C
14Chronoamperometric Response
- Substrate Sorbitol
- Concentration 5 mM
- Kinetic parameters
- k 690 s-1
- k 87 s-1
- Surface coverage
- ? 8.7?10-12 mol cm-2
- ? 8.0?10-12 mol cm-2
15Cyclic Voltammetric Response
- Concentration range 3-21 mM
- Sensitivity 3.4 mA mM-1 cm-2
- TRmax38 s-1
16Amperometric Response
- Potential -200 mV
- Concentration range 1-6 mM
- Sensitivity 2.8 mA mM-1 cm-2
17Other Enzymes Used
- Mannitol dehydrogenase
- Organism Lactobacillus reuteri
- Reaction Fructose Mannitol
- Cofactor specificity NAD
- Thermal stability 50?C-90?C
18Other Enzymes Used
- Secondary alcohol dehydrogenase
- Organism Thermoanaerobacter ethanolicus
- Reaction 2-Propanol
Acetone - Cofactor specificity NADP
- Thermal stability 30?C-100?C
19Chronoamperometric Results
Chronoamperometric measurements were made at a
concentration of 5 mM of the substrate.
20Cyclic Voltammetry Results
21Conclusions
- Developed self-assembling biosensor array
- Multiple analyte detection
- Sorbitol
- Mannitol
- 2-Propanol
- Characterized interfaces electrochemically
- Chronoamperometry
- Cyclic voltammetry
- Constant potential amperometry
22Acknowledgments-
- Ted Amundsen (CHEMS-MSU)
- Yue Huang (EECS-MSU)
- Kikkoman Corporation
- Funding sources
- Michigan Technology Tri-Corridor (MTTC)
- IRGP programs at MSU
- Department of Education GAANN Fellowship
23Thank you
24Reaction Mechanism
Hassler et. al, Biosensors and Bioelectronics,
77, 4726-4733 (2006)
25Secondary Alcohol Dehydrogenase (2? ADH)
- Organism Thermoanaerobacter ethanolicus
- Cofactor dependence NADP
- Temperature stability 30-100?C
26Mannitol Dehydrogenase
- Organism Lactrobacillus reuteri
- Cofactor dependence NAD
- Temperature stability 50-80?C
27Chronoamperometry response with MDH
- Electron transfer coefficients
- ket 5.0102 s-1
- ket 4.5101 s-1
- Surface coverage
- ? 7.210-12 mol cm-2
- ? 6.010-12 mol cm-2
28Chronoamperometry response with 2? ADH
- Electron transfer coefficients
- ket 6.9102 s-1
- Surface coverage
- ? 1.610-11 mol cm-2